Transformed soybean plant which accumulates vaccine, and use thereof

ABSTRACT

A transformed soybean plant having a gene encoding a modified seed storage protein introduced therein, obtained by inserting a gene encoding an Alzheimer&#39;s disease vaccine to a variable region(s) of a gene encoding a wild-type seed storage protein, is produced, and said vaccine is produced and accumulated in the seeds thereof.

TECHNICAL FIELD

The present invention relates to a transformed soybean plant whichaccumulates an Alzheimer's disease vaccine in its seeds, and usethereof.

BACKGROUND ART

Alzheimer's disease is a neurodegenerative disease caused byaccumulation of a causative substance such as β-amyloid in brain,causing damage to nerve cells. Although the number of patients sufferingfrom Alzheimer's disease is expected to increase upon the advent of anaging society, prophylactic agents and therapeutic agents for thedisease are hardly available, and development of new prophylacticagents, therapeutic agents, vaccines and the like has been demanded.Vaccines against Alzheimer's disease have been developed usingβ-amyloid, which is a causative substance of the disease, as an antigen,but development of the vaccines was difficult because of problems suchas side effects. Therefore, development of a vaccine using a β-amyloidantigenic determinant (epitope) which does not cause side effects, andestablishment of mass production techniques for the vaccine arerequired.

Soybean is an exalbuminous seed, which does not have albumen andaccumulates its nutrition in the germ corresponding to the cotyledon.About 40% of the whole volume of a seed, which corresponds to the germ,is occupied by storage proteins. Therefore, soybean has characteristicsas a storage tissue different from those of other crops such as rice andmaize that accumulate starch in albumen as a major reserve substance, sothat it is a crop suitable for being made to produce and accumulate anexogenous protein. The major seed storage proteins in soybean are 11Sglobulin (glycinin) and 7S globulin (β-conglycinin). The spatialstructures of these seed storage proteins and the mechanisms of theiraccumulation in the cell have been elucidated, and it is known that thegenes encoding them have portions called variable regions. It is thoughtthat the spatial structures of the proteins can be maintained even afterinsertion of an exogenous gene into the variable regions and that theproperties of the storage proteins are not affected by such insertion.

In general, a β-amyloid antigenic determinant is a protein (peptide)having a relatively low molecular weight composed of several aminoacids, and it has been difficult to make the peptide highly accumulatedin seeds of a transformed soybean for the purpose of mass production ofthe peptide by introducing a gene encoding the peptide to the soybean,since the peptide was degraded by enzymes such as proteases in thecells.

On the other hand, as transformed crops that accumulate biologicallyactive peptides and vaccines in their seeds, a transformed soybean thataccumulates a hypotensive peptide (Patent Document 1), a transformedrice that accumulates a vaccine against allergy to cedar pollen (PatentDocument 2), a potato that produces β-amyloid (Non-patent Document 1)and a tomato that produces β-amyloid (Non-patent Document 2) are known.

However, a transformed soybean that highly accumulates an Alzheimer'sdisease vaccine composed of a β-amyloid antigenic determinant (epitope),and mass production techniques for the vaccine using the soybean havenot been known so far.

Common bean is a plant belonging to Leguminosae, to which soybean alsobelongs, and the content of protein in a seed of common bean is 20%. Itis known that arcelin, which is one of the major seed storage proteinsin common bean, can be divided into plural types, that is, arcelin 1 to7, and that the homologies among the nucleotide sequences of the partencoding their structural proteins are high. The structures of thearcelin proteins in common bean have been less analyzed compared tothose in soybean, and only the spatial structures of arcelin 1 and 5have been revealed.

Further, it is known that prolamin, which is one of the major seedstorage proteins in rice, is an indigestible protein which can bedivided into several types (e.g., 10K, 13K and 16K) having differentmolecular weights. The spatial structure of prolamin has not beenrevealed.

PRIOR ART DOCUMENTS Patent Documents

Patent Document 1: JP 2006-238821 A

Patent Document 2: JP 2004-321079 A

Non-Patent Documents

Non-patent Document 1: Federation of European Biochemical Societies(2005) vol. 579, pp. 6737-6744.

Non-patent Document 2: Biotechnology Letters (2008) vol. 30, pp.1839-1845.

DISCLOSURE OF THE INVENTION

The present invention aims to provide a transformed soybean plant whichcan be made to produce and accumulate an Alzheimer's disease vaccine inits seeds. Further, the present invention aims to provide a method forproducing an Alzheimer's disease vaccine using the transformed soybean.

The present inventors intensively studied to solve the above problems.

As a result, the present inventors succeeded in preparation of atransformed soybean plant having a gene encoding a modified seed storageprotein introduced therein, which gene has been obtained by inserting agene encoding an Alzheimer's disease vaccine to a variable region(s) ofa gene encoding a wild-type seed storage protein, and also succeeded inproduction and accumulation of the Alzheimer's disease vaccine in seedsof the transformed soybean plant.

Further, the present inventors discovered that a transformed soybeanplant produced by introducing the gene encoding a modified seed storageprotein to soybean in which an endogenous seed storage protein(s) is/aredeficient can efficiently produce and accumulate the Alzheimer's diseasevaccine in its seeds.

That is, the present invention provides:

-   [1] A transformed soybean plant having an introduced gene encoding a    modified seed storage protein, which gene encoding a modified seed    storage protein was produced by inserting a gene encoding an    Alzheimer's disease vaccine to a variable region(s) of a gene    encoding a wild-type seed storage protein such that frameshift does    not occur, which modified seed storage protein is expressed in a    seed and accumulates therein.-   [2] The transformed soybean plant according to [1], wherein the    Alzheimer's disease vaccine is a β-amyloid antigenic determinant.-   [3] The transformed soybean plant according to [2], wherein the    β-amyloid antigenic determinant has a sequence having one to three    copies of the peptide having the sequence shown in SEQ ID NO:3 which    are linked to each other.-   [4] The transformed soybean plant according to any one of [1] to    [3], wherein endogenous soybean 11S globulin and/or soybean 7S    globulin is/are deficient.-   [5] The transformed soybean plant according to any one of [1] to    [4], wherein the wild-type seed storage protein is the A1aB1b    subunit of soybean 11S globulin, arcelin of common bean, or prolamin    of rice.-   [6] The transformed soybean plant according to [5], wherein the    wild-type seed storage protein contains the amino acid sequence    shown in SEQ ID NO:2 or an amino acid sequence having an identity of    not less than 90% to the amino acid sequence shown in SEQ ID NO:2,    and the variable region(s) to which the gene encoding an Alzheimer's    disease vaccine was inserted is/are the region(s) encoding one or    more amino acid sequence(s) selected from the group consisting of    the amino acid sequences corresponding to amino acid positions    111-128, amino acid positions 198-216, amino acid positions 268-315    and amino acid positions 490-495 in SEQ ID NO:2.-   [7] The transformed soybean plant according to [5], wherein the    wild-type seed storage protein contains the amino acid sequence    shown in SEQ ID NO:37 or an amino acid sequence having an identity    of not less than 90% to the amino acid sequence shown in SEQ ID    NO:37, and the variable region(s) to which the gene encoding an    Alzheimer's disease vaccine was inserted is/are the region(s)    encoding the amino acid sequence(s) corresponding to amino acid    positions 149-150 and/or amino acid positions 250-251 in SEQ ID    NO:37.-   [8] The transformed soybean plant according to [5], wherein the    wild-type seed storage protein contains the amino acid sequence    shown in SEQ ID NO:45 or an amino acid sequence having an identity    of not less than 90% to the amino acid sequence shown in SEQ ID    NO:45, and the variable region to which the gene encoding an    Alzheimer's disease vaccine was inserted is the region encoding the    amino acid sequence corresponding to amino acid positions 110-111 in    SEQ ID NO:45.-   [9] The transformed soybean plant according to any one of [1] to    [8], wherein expression of the gene encoding a modified seed storage    protein is regulated by the promoter of common bean arcelin 2 or the    promoter of the A1aB1b subunit of soybean 11S globulin.-   [10] A cell of the transformed soybean plant according to any one of    [1] to [9].-   [11] A seed of the transformed soybean plant according to any one of    [1] to [9].-   [12] A processed soybean seed produced by processing the seed    according to [11].-   [13] A method for producing an Alzheimer's disease vaccine using the    transformed soybean plant according to any one of [1] to [9], which    Alzheimer's disease vaccine is produced in a seed of the transformed    soybean plant.-   [14] A vector comprising:

a promoter which induces soybean seed-specific expression; and

a gene encoding a modified seed storage protein produced by inserting agene encoding an Alzheimer's disease vaccine to a variable region(s) ofa gene encoding a wild-type seed storage protein such that frameshiftdoes not occur, which gene encoding a modified seed storage protein islinked to the downstream of the promoter.

-   [15] The vector according to [14], wherein the promoter is the    promoter of common bean arcelin 2 or the promoter of the A1aB1b    subunit of soybean 11S globulin.

Since the transformed soybean plant of the present invention can highlyaccumulate an Alzheimer's disease vaccine in its seeds, the Alzheimer'sdisease vaccine can be efficiently produced using the transformedsoybean plant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing the amino acid sequences encodedby the variable regions in the amino acid sequence of the A1aB1b subunitof soybean glycinin.

FIG. 2 is a diagram showing the construction procedure for a plasmidpUHGA1aB1bM.

FIG. 3 is a schematic diagram showing the structure of the plasmid pUHGwhich was used for incorporation of a gene encoding a modified seedstorage protein. The gene of an arbitrary modified seed storage proteinis inserted into the SmaI site.

FIG. 4 shows photographs showing detection of accumulation of aβ-amyloid antigenic determinant in seeds of a transformed soybean towhich a gene encoding a modified A1aB1M1 was introduced, which detectionwas carried out by Western blotting. The samples in the respective lanesare as follows. 1: Transformed soybean 10-2 No. 1 produced byintroducing A1aB1M1 to variety Jack; 2: transformed soybean 10-2 No. 2produced by introducing A1aB1M1 to variety Jack; 3: transformed soybean10-2 No. 3 produced by introducing A1aB1M1 to variety Jack; 4: varietyJack (control); 5: transformed soybean 16-2 No. 1 produced byintroducing A1aB1M1 to a storage protein-deficient line; 6: transformedsoybean 16-2 No. 2 produced by introducing A1aB1M1 to a storageprotein-deficient line; 7: transformed soybean 16-2 No. 3 produced byintroducing A1aB1M1 to a storage protein-deficient line; and 8: astorage protein-deficient line (control).

FIG. 5 shows photographs showing detection of accumulation of β-amyloidantigenic determinant in seeds of a transformed soybean to which a geneencoding modified arcelin (Arc5M1) was introduced, which detection wascarried out by Western blotting. The samples in the respective lanes areas follows. 1: Transformed soybean 2-1 No. 1 produced by introducingArc5M1 to variety Jack; 2: transformed soybean 2-1 No. 2 produced byintroducing Arc5M1 to variety Jack; 3: transformed soybean 2-2 No. 1produced by introducing Arc5M1 to variety Jack; and 4: transformedsoybean 2-2 No. 2 produced by introducing Arc5M1 to variety Jack.

FIG. 6 shows a photograph showing detection of accumulation of aβ-amyloid antigenic determinant in seeds of a transformed soybean towhich a gene encoding modified prolamin (PR10M1) was introduced, whichdetection was carried out by Western blotting. The samples in therespective lanes are as follows. 1: Transformed soybean 1-1 No. 1produced by introducing PR10M1 to variety Jack; 2: transformed soybean1-1 No. 2 produced by introducing PR10M1 to variety Jack; 3: transformedsoybean 4-2 No. 1 produced by introducing PR10M1 to variety Jack; and 4:transformed soybean 4-2 No. 2 produced by introducing PR10M1 to varietyJack.

FIG. 7 shows photographs showing detection of accumulation of aβ-amyloid antigenic determinant in seeds of a transformed soybean towhich a gene encoding modified A1aB1M1 or A2PA1aB1bM3 was introduced,which detection was carried out by Western blotting. The samples in therespective lanes are as follows. 1: transformed soybean line 4-6produced by introducing A2PA1aB1bM1 to a storage protein-deficient line;2: transformed soybean line 7-1 produced by introducing A1aB1bM1 to astorage protein-deficient line; 3: transformed soybean line 8-1 No. 1produced by introducing A1aB1bM1 to variety Jack; 4: transformed soybeanline 8-1 No. 2 produced by introducing A1aB1bM1 to variety Jack; 5:storage protein-deficient line; 6: transformed soybean line 3-1 No. 1produced by introducing A2PA1aB1bM3 to a storage protein-deficient line;and 7: transformed soybean line 3-1 No. 2 produced by introducingA2PA1aB1bM3 to a storage protein-deficient line.

FIG. 8 shows a diagram (photographs) showing the results of a stabilityassay of Aβ4-10 (the peptide having the amino acid sequence shown in SEQID NO:3) in seeds of the transformed soybean A1aB1bM3. The samples inthe respective lanes are as follows. A-1, A-2: heat-untreated group;B-1, B-2: roasted group; C-1, C-2: water-boiled group; D-1, D-2:heat-treated extract group.

FIG. 9 shows a diagram (photographs) showing evaluation of therelationship between the repeat number of Aβ4-10 and the antibody titer.The samples in the respective lanes are as follows. 1: Reaction group inwhich purified antibody key-limpet-hemocyanin (KLH)-P1 was allowed toreact with substrate Aβ2 (400 picomoles); 2: reaction group in whichpurified antibody KLH-P1 was allowed to react with substrate Aβ42 (1000picomoles); 3: reaction group in which purified antibody KLH-P2 wasallowed to react with substrate Aβ42 (400 picomoles); 4: reaction groupin which purified antibody KLH-P2 was allowed to react with substrateAβ42 (1000 picomoles); 5: reaction group in which purified antibodyKLH-P3 was allowed to react with substrate Aβ42 (400 picomoles); and 6:reaction group in which purified antibody KLH-P3 was allowed to reactwith substrate Aβ42 (1000 picomoles).

MODES FOR CARRYING OUT THE INVENTION

The present invention will now be described in detail.

1. Gene Encoding Wild-Type Seed Storage Protein

Examples of the wild-type seed storage protein in the present inventioninclude the respective subunits constituting soybean 11S globulin, therespective subunits constituting soybean 7S globulin, arcelin in commonbean, prolamin in rice, globulin in rice, and further, seed storageproteins in other crops. Preferred examples of the wild-type seedstorage protein include the A1aB1b subunit of 11S globulin and the asubunit and β subunit of 7S globulin in soybean, among which the A1aB1bsubunit of 11S globulin in soybean is more preferred.

Further, examples of the wild-type seed storage protein in the presentinvention include proteins containing the amino acid sequences shown inSEQ ID NO:2, SEQ ID NO:37 and SEQ ID NO:45, and proteins containingamino acid sequences having identities of not less than 80%, preferablynot less than 90%, more preferably not less than 95% to these amino acidsequences.

Here, the identity (%) between amino acid sequences means the maximumidentity (%) between the amino acid sequences which is obtained byaligning the two amino acid sequences to be compared while introducing,as required, gaps thereto (alignment). The alignment for the purpose ofdetermining the identity between amino acid sequences can be carried outusing various methods which are well-known to those skilled in the art.For example, publicly available computer software such as BLAST,BLAST-2, ALIGN and Megalign (DNASTAR) software and commerciallyavailable software such as Gene Works 2.5.1 software (Teijin SystemTechnology, Inc.) and GENETIX-WIN (Software Development Co., Ltd) may beused.

Examples of the gene encoding a wild-type seed storage protein in thepresent invention include genes encoding the respective subunitsconstituting soybean 11S globulin, genes encoding the respectivesubunits constituting soybean 7S globulin, a gene encoding arcelin incommon bean, a gene encoding prolamin in rice, a gene encoding globulinin rice, and further, genes encoding seed storage proteins in othercrops. Preferred examples of the gene include a gene encoding the A1aB1bsubunit of 11S globulin and genes encoding the α subunit and β subunitof 7S globulin in soybean, among which a gene encoding the A1aB1bsubunit of 11S globulin in soybean is more preferred.

Further, examples of the gene encoding a wild-type seed storage proteinin the present invention include genes containing the nucleotidesequences shown in SEQ ID NO:1, SEQ ID NO:36 and SEQ ID NO:44, and genescontaining nucleotide sequences having identities of not less than 80%,preferably not less than 90%, more preferably not less than 95% to thesenucleotide sequences.

Here, the identity (%) between nucleotide sequences means the maximumidentity (%) between the nucleotide sequences which is obtained byaligning the two nucleotide sequences to be compared while introducing,as required, gaps thereto (alignment). The alignment for the purpose ofdetermining the identity between nucleotide sequences can be carried outusing various methods which are well-known to those skilled in the art.For example, publicly available computer software such as BLAST,BLAST-2, ALIGN and Megalign (DNASTAR) software and commerciallyavailable software such as Gene Works 2.5.1 software (Teijin SystemTechnology, Inc.) and GENETIX-WIN (Software Development Co., Ltd) may beused.

2. Gene Encoding Modified Seed Storage Protein

The gene encoding a modified seed storage protein in the presentinvention means a gene produced by inserting a gene encoding anAlzheimer's disease vaccine to a variable region(s) of a gene encoding awild-type seed storage protein such that frameshift does not occur.

Here, “inserting a gene encoding an Alzheimer's disease vaccine suchthat frameshift does not occur” means that the gene encoding anAlzheimer's disease vaccine has been inserted to a variable region(s) ofa gene encoding a wild-type seed storage protein such that the aminoacid sequence of the modified seed storage protein excluding the aminoacid sequence of the Alzheimer's disease vaccine is identical to theamino acid sequence of the corresponding wild-type seed storage protein.

Further, “inserting a gene encoding an Alzheimer's disease vaccine”means that the gene encoding an Alzheimer's disease vaccine has beeninserted without causing deletion of a nucleotide sequence encoding thevariable region(s), as well as that all or a part of the nucleotidesequence encoding the variable region(s) was substituted with the geneencoding an Alzheimer's disease vaccine.

In the present invention, a variable region of a gene encoding awild-type seed storage protein means a region which allows, even when anexogenous gene has been inserted thereto such that frameshift does notoccur, the protein expressed from the resulting gene to maintain astable spatial structure equivalent to that of the wild-type seedstorage protein, thereby allowing maintenance of the properties of thewild-type seed storage protein.

For example, when the gene encoding a wild-type seed storage protein isthe gene encoding the A1aB1b subunit of soybean 11S globulin containingthe amino acid sequence of SEQ ID NO:2, five portions including theregion encoding amino acid positions 20-28 (variable region I), theregion encoding amino acid positions 111-128 (variable region II), theregion encoding amino acid positions 198-216 (variable region III), theregion encoding amino acid positions 268-315 (variable region IV) andthe region encoding amino acid positions 490-495 (variable region V) inSEQ ID NO:2 are known as variable regions (FIG. 1).

Further, when the gene encoding a wild-type seed storage protein is agene containing a nucleotide sequence encoding an amino acid sequencewhich has a certain identity to the amino acid sequence shown in SEQ IDNO:2, that is, the amino acid sequence shown in SEQ ID NO:2 except thatone or more amino acids are substituted, inserted, added and/or deleted,the variable regions are the region encoding the amino acid sequencecorresponding to amino acid positions 20-28, the region encoding theamino acid sequence corresponding to amino acid positions 111-128, theregion encoding the amino acid sequence corresponding to amino acidpositions 198-216, the region encoding the amino acid sequencecorresponding to amino acid positions 268-315 and the region encodingthe amino acid sequence corresponding to amino acid positions 490-495.

Here, when two amino acid sequences to be compared are aligned with eachother to attain the maximum identity (%) between the amino acidsequences while introducing gaps as required, the “amino acid sequencecorresponding to” a particular amino acid sequence means a partial aminoacid sequence that corresponds to the other particular partial aminoacid sequence. Such an amino acid sequence can be easily specified bythose skilled in the art.

When plural variable regions exist in the gene encoding a wild-type seedstorage protein, the gene encoding a modified seed storage protein canbe prepared by inserting a gene encoding an Alzheimer's disease vaccineto one or more of the variable regions.

For example, when a gene encoding the A1aB1b subunit of soybean 11Sglobulin containing the amino acid sequence of SEQ ID NO:2 is used asthe gene encoding a wild-type seed storage protein, any one of thevariable regions II, III, IV and V may be selected as the variableregion to which the gene encoding an Alzheimer's disease vaccine is tobe inserted, and the gene encoding an Alzheimer's disease vaccine ismore preferably inserted to the variable region III. Further, as thevariable regions to which the gene encoding an Alzheimer's diseasevaccine is to be inserted, two or more regions among the variableregions II, III, IV and V may be selected, and, for example, insertioninto the three regions II, III and IV at the same time, insertion intothe four regions II, III, IV and V at the same time, and the like can becarried out. Here, introduction of the gene encoding an Alzheimer'sdisease vaccine needs to be carried out such that frameshift does notoccur in the nucleotide sequence encoding the wild-type seed storageprotein.

Further, when a gene encoding common bean arcelin 5 containing the aminoacid sequence shown in SEQ ID NO:37 is used as the gene encoding awild-type seed storage protein, since the variable region(s) of the geneis/are not known, it is necessary to compare its DNA sequence with thatof the A1aB1b subunit to confirm disordered regions, and to compare itsamino acid sequence and spatial structure with those of other similarstorage proteins to confirm the differences in the gaps of the sequenceand the structural differences, thereby assuming the variable region(s).It is preferred to insert the gene encoding an Alzheimer's diseasevaccine into the region encoding amino acid positions 149-150 (variableregion A) and/or the region encoding amino acid positions 250-251(variable region B) in SEQ ID NO:37, which regions can be specified bysuch assumption.

Further, when the gene encoding the wild-type seed storage proteincontains a nucleotide sequence encoding an amino acid which has acertain identity to the amino acid sequence shown in SEQ ID NO:37, thatis, the amino acid sequence shown in SEQ ID NO:37 except that one ormore amino acids are substituted, inserted, added and/or deleted, thegene encoding an Alzheimer's disease vaccine is preferably inserted intothe region encoding the amino acid sequence corresponding to amino acidpositions 149-150, the region encoding the amino acid sequencecorresponding to amino acid positions 250-251 in SEQ ID NO:37.

Further, when a gene encoding rice prolamin containing the amino acidsequence shown in SEQ ID NO:45 is used as the gene encoding a wild-typeseed storage protein, since the spatial structure and the variableregion(s) of the gene are not known, it is necessary to compare itsamino acid sequence with those of other similar storage proteins toconfirm the gap structure, thereby assuming the variable region(s). Itis preferred to insert the gene encoding an Alzheimer's disease vaccineinto the region encoding amino acid positions 110-111 (variable regiona) in SEQ ID NO:45, which region can be specified by such assumption.

Further, when the gene encoding the wild-type seed storage proteincontains a nucleotide sequence encoding an amino acid which has acertain identity to the amino acid sequence shown in SEQ ID NO:45, thatis, the amino acid sequence shown in SEQ ID NO:45 except that one ormore amino acids are substituted, inserted, added and/or deleted, thegene encoding an Alzheimer's disease vaccine is preferably inserted intothe region encoding the amino acid sequence corresponding to amino acidpositions 110-111 in SEQ ID NO:45.

3.Gene Encoding Alzheimer's Disease Vaccine

The gene encoding an Alzheimer's disease vaccine in the presentinvention is not restricted as long as it is a DNA encoding a protein ora peptide having a function as a vaccine against Alzheimer's disease,and is preferably a DNA encoding a β-amyloid antigenic determinantcomposed of a peptide of about 5 to 25 amino acids constituting a partof β-amyloid. Examples of the DNA include DNAs encoding the amino acidsequence of SEQ ID NO:3.

Since a nucleotide sequence encoding β-amyloid is known (GenBankaccession No. AB113349), it is possible to isolate a DNA encodingβ-amyloid or a β-amyloid antigenic determinant from a cDNA library, by ascreening operation based on this nucleotide sequence information. A DNAencoding a β-amyloid antigenic determinant can be prepared also bychemical synthesis.

Further, in the present invention, the Alzheimer's disease vaccine canalso be inserted to a variable region(s) of a gene encoding a seedstorage protein in such a manner that plural genes encoding the vaccineare tandemly linked to each other, thereby allowing expression of thevaccine. For example, the gene encoding a β-amyloid antigenicdeterminant may be inserted, such that frameshift does not occur, to avariable region(s) in such a manner that an integer number of 1 to 20,preferably an integer number of 1 to 5, more preferably an integernumber of 1 to 3, especially preferably copies of the gene are linked toeach other.

Further, when the gene encoding an Alzheimer's disease vaccine isinserted into the gene encoding a wild-type seed storage protein, anucleotide sequence(s) which encode(s) a sequence recognized by aprotease may also be added to the 5′-end and/or the 3′-end of the geneencoding an Alzheimer's disease vaccine. By this, the Alzheimer'sdisease vaccine can be cleaved out with the protease from the modifiedseed storage protein produced in seeds. Examples of such a proteaseinclude thermolysin.

4. Vector for Gene Transfer

The vector for gene transfer of the present invention may have astructure wherein a promoter that induces soybean seed-specificexpression is linked to the upstream of the gene. Further, a terminatormay also be linked to the downstream of the gene.

Examples of the promoter that induces soybean seed-specific expressioninclude the soybean 11S globulin promoter and the common bean arcelinpromoter, and examples of the terminator include the soybean 11Sglobulin terminator, the common bean arcelin 2 terminator, the 35Sterminator and the NOS terminator of cauliflower mosaic virus.

Examples of the soybean 11S globulin promoter include the promoter ofthe soybean 11S globulin A1aB1b subunit having the sequence shown in SEQID NO:18, and the soybean 11S globulin promoter may be one having anidentity of not less than 95% to this sequence as long as it has aseed-specific promoter activity. Examples of the soybean 11S globulinterminator include the terminator of the soybean 11S globulin A1aB1bsubunit having the sequence shown in SEQ ID NO:21, and the soybean 11Sglobulin terminator may be one having an identity of not less than 95%to this sequence as long as it has a seed-specific terminator activity.

Examples of the common bean arcelin promoter include the promoter ofcommon bean arcelin 2 having the sequence of nucleotide positions1399-3860 in SEQ ID NO:56, and the common bean arcelin promoter may beone having an identity of not less than 95% to this sequence as long asit has a seed-specific promoter activity. Examples of the common beanarcelin terminator include the common bean arcelin 2 terminator havingthe sequence shown in SEQ ID NO:59, and the common bean arcelinterminator may be one having an identity of not less than 95% to thissequence as long as it has a seed-specific terminator activity.

To the vector of the present invention, a selection marker gene forselecting recombinants, and a reporter gene for confirming expression ofthe introduced gene may be inserted. Examples of the selection markergene include the hygromycin resistance gene, the phosphinothricinresistance gene or the like, and examples of the reporter gene includethe β-glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT)gene, luciferase (LUC) gene and GFP gene or the like.

The vector of the present invention may be obtained also by inserting aDNA fragment, which contains a promoter for induction of seed-specificexpression, a gene encoding a modified storage protein linked to thedownstream of the promoter, and a terminator linked to the furtherdownstream, to a vector comprising the above-described selection markergene and/or reporter gene.

5. Soybean Plant Which Can Be Transformed

Examples of the soybean which can be transformed in the presentinvention include varieties which are generally used for food, for feedor for producing oil. The soybean to be transformed is preferablypartially or totally deficient for the endogenous seed storage proteins,and examples of the soybean include those which are partially or totallydeficient for soybean 11S globulin and/or soybean 7S globulin. Here,“partially deficient” means cases where the expression level is lowerthan in the wild type, as well as cases where only a part of thesubunits are completely deficient, and cases where the expression levelsof only a part of the subunits are lower than those in the wild type.Particular examples of the soybean include the mutant line EnB1, whichis deficient for soybean 11S globulin, the mutant line QY2, which isdeficient for soybean 7S globulin, and the mutant line QF2, which isdeficient for both 11S globulin and 7S globulin. Further examples of thesoybean include progeny lines derived by hybridization between suchdefective lines and common varieties (e.g., Jack or the like).

Confirmation of the fact that the soybean is partially or totallydeficient for endogenous soybean 11S globulin and/or soybean 7S globulincan be carried out by electrophoresis of the storage proteins preparedfrom seeds.

6. Preparation of Transformed Soybean Plant

Examples of the material which may be used for preparation of thetransformed soybean plant include plant tissues such as roots, stems,leaves, seeds, embryos, ovules, ovaries, shoot apices, anther andpollens, and sections thereof; and plant cultured cells such asundifferentiated calluses, adventive embryos and protoplasts or thelike.

The introduction of the gene encoding a modified seed storage protein tothe above material may be carried out by various methods which havealready been reported and established, and it is preferred to introducethe above-mentioned vector for gene transfer using the Agrobacteriummethod, PEG method, electroporation method, particle gun method, whiskerultrasonic method or the like.

Using the resistance effect given by the selection marker gene as anindex, cells of the transformed soybean plant can be selected from thematerial to which the gene encoding a modified seed storage protein hasbeen introduced. From the selected cells, a transformed soybean plantbody can be obtained through the step of regenerating a plant body,which step has been reported for each plant species.

By cultivating the thus obtained transformed soybean plant body to allowseed ripening, seeds of the transformed soybean of the present inventioncan be obtained, and the Alzheimer's disease vaccine of interest can beobtained in the transformed seeds.

Whether or not the gene encoding the Alzheimer's disease vaccine hasbeen introduced into the plant body can be confirmed by the PCR method,Southern hybridization method, Northern hybridization method, Westernblotting method or the like. For example, by extracting protein fromseeds of the transformed soybean plant body and carrying out Westernblotting by immunostaining using a primary antibody specific to theAlzheimer's disease vaccine and a secondary antibody labeled withhorseradish peroxidase (HRP) or the like, it is possible to confirmappropriate introduction of the gene encoding the Alzheimer's diseasevaccine, accumulation of the Alzheimer's disease vaccine in the seeds,and the amount of the vaccine accumulated.

The performance of the Alzheimer's disease vaccine contained in themodified seed storage protein accumulated in the seeds of thetransformed soybean plant can be evaluated by, for example, using adisease-model mouse that develops Alzheimer's disease. Moreparticularly, the modified seed storage protein containing theAlzheimer's disease vaccine, or the Alzheimer's disease vaccine cleavedout from the modified seed storage protein with a protease followed bypurification, is administered to the above model mouse by subcutaneousinjection or oral administration. The performance of the Alzheimer'sdisease vaccine can be evaluated by investigating production ofantibodies against the Alzheimer's disease vaccine, the amount ofβ-amyloid, brain tissue, and/or behavior disorder in the mouse. Themodified seed storage protein containing the Alzheimer's diseasevaccine, or the Alzheimer's disease vaccine cleaved out from themodified seed storage protein with a protease followed by purification,may be administered as a mixture with an adjuvant.

The Alzheimer's disease vaccine can be produced in a large amount bycultivating and then collecting seeds of the transformed soybean thataccumulates a modified seed storage protein containing the vaccine, inthe outdoor field, or closed facilities for cultivation where theenvironment is artificially controlled.

The seeds wherein the modified seed storage protein containing theAlzheimer's disease vaccine is accumulated can be used for prophylaxisand/or therapy of Alzheimer's disease, as a composition containing theAlzheimer's disease vaccine. For example, the seeds processed bypulverization or the like may be made into the form of a tablet,granule, powder, capsule, beverage or the like.

Further, the above composition may contain the modified seed storageprotein accumulated in the seeds, which protein has been extracted andpurified. For example, after a ground product of the seeds subjected todefatting and heat treatment, the modified seed storage proteincontaining the Alzheimer's disease vaccine of interest may be purifiedby an apparatus such as liquid chromatography. Further, theabove-described composition may contain the Alzheimer's disease vaccinewhich has been prepared by treating the modified seed storage proteinwith a protease and purifying the resulting product, thereby partiallyor totally removing the part of the wild-type seed storage protein fromthe modified seed storage protein.

The present invention will now be described more concretely by way ofExamples, but the present invention is not restricted to these Examples.

The procedures of the experimental methods carried out in the Examplesbelow are those according to “Molecular Cloning” 2nd Ed. (J. Sambrook etal., Cold Spring Harbor Laboratory press, published in 1989) unlessotherwise specified.

EXAMPLES Example 1

Construction of Expression Plasmids for Modified Soybean 11S GlobulinA1aB1b

Expression plasmids for expression of genes encoding modified A1aB1bcontaining the peptide having the amino acid sequence shown in SEQ IDNO:3 (hereinafter abbreviated as Aβ4-10), which is known as a β-amyloidantigenic determinant, in soybean seeds were constructed. The procedurefor the construction is shown in FIG. 2.

An oligonucleotide having three copies of a nucleotide sequence encodingAβ4-10 which are tandemly linked to each other (the sense strand, SEQ IDNO:4) and the oligonucleotide having its complementary sequence (theantisense strand, SEQ ID NO:5) were synthesized using the custom DNAsynthesis service by FASMAC Co., Ltd. (the sense strand and theantisense strand are hereinafter referred to as 410F and 410R,respectively). Unless otherwise specified, the hereinafter-mentionedoligonucleotides were those synthesized using the custom DNA synthesisservice by the above manufacturer. In the presence of ATP at a finalconcentration of 1 mM, 100 pmol each of 410F and 410R was subjected tophosphorylation reaction with T4 Polynucleotide Kinase (manufactured byTAKARA BIO INC.), and the reaction solutions after the reaction weremixed together, followed by heating the resulting mixture at 94° C. for10 minutes and then allowing the mixture to cool gradually to 37° C. for1 hour, thereby carrying out annealing. By this process, adouble-stranded DNA fragment encoding a peptide wherein three copies ofAβ4-10 are tandemly linked to each other ((Aβ4-10)×3) was obtained.

Using, as a template, the plasmid pBSK-A1aB1b (obtained from KyotoUniversity) wherein cDNA of the known A1aB1b gene (GenBank accession No.AB113349) is cloned at the SmaI site of pBluescript II SK(−)(manufactured by Stratagene), PCR was carried out to amplify a fragmentcontaining the vector portion such that the 5′-end and the 3′-end of thefragment are positioned at a specific variable region of the geneencoding A1aB1b. The obtained DNA fragment was ligated with thedouble-stranded DNA fragment encoding (Aβ4-10)×3, to prepare the geneencoding a modified A1aB1b. The method is more concretely describedbelow.

A total of five primer sets, that is, the primer set (PS-1) composed ofthe primer pair of SEQ ID NOs:6 and 7 for insertion into the variableregion II, the primer set (PS-2) composed of the primer pair of SEQ IDNOs:8 and 9 for insertion into the variable region III, the primer set(PS-3) composed of the primer pair of SEQ ID NOs:10 and 11 and theprimer set (PS-4) composed of the primer pair of SEQ ID NOs:12 and 13for insertion into the variable region IV, and the primer set (PS-5)composed of the primer pair of SEQ ID NOs:14 and 15 for insertion intothe variable region V, of the gene encoding A1aB1b having the sequenceshown in SEQ ID NO:1 were prepared. The primers were synthesized suchthat nucleotide substitutions for introducing amino acid substitutionsin the immediate downstream of the insertion regions were introduced inorder to allow cleaving out of (Aβ4-10)×3 from the modified A1aB1bproteins using a protease thermolysin.

The regions in the nucleotide sequence shown in SEQ ID NO:1, into whichthe DNA encoding (Aβ4-10)×3 was inserted using the respective primersets are hereinafter referred to as the PS-1 region, PS-2 region, PS-3region, PS-4 region and PS-5 region, respectively.

PCR was performed using 10 ng of pBSK-A1aB1b as a template and 50μL/reaction of a reaction solution, by carrying out 1 cycle of 2 minutesof denaturation at 94° C. and then 25 cycles of 30 seconds ofdenaturation at 94° C., 30 seconds of annealing at 57° C. and 5 minutesof extension at 68° C. The reaction solution contained 200 μM dNTPmixture, 1.5 mM MgSO₄ solution, each of the above primers at aconcentration of 1 μM, and KOD-Plus-Ver.2 buffer containing 1 unit ofKOD-Plus-(manufactured by Toyobo Co. Ltd.). The hereinafter-mentionedPCRs were carried out using the same composition except for the primers,unless otherwise specified.

Using DNA Ligation Kit (manufactured by TAKARA BIO), 50 fmol of each ofthe thus obtained DNA fragments and 150 fmol of the double-stranded DNAfragment encoding the above-described (Aβ4-10)×3 were subjected toligation reaction at 16° C. for 40 minutes. The reaction product wasused for transformation of E. coli DH5α competent cells (manufactured byTAKARA BIO) to obtain plural transformed E. coli cells. From theobtained E. coli cells, plasmid DNAs were extracted and purified,followed by analyzing their nucleotide sequences using the DNAsequencing service by FASMAC Co., Ltd. In the case where PS-1 was used,the result of the nucleotide sequence analysis of 12 clones of thetransformed E. coli showed that one clone had one molecule of thedouble-stranded DNA fragment encoding (Aβ4-10)×3 in a state where thefragment was correctly inserted in the forward direction. Further, 24clones, 54 clones, 30 clones and 12 clones were analyzed in the caseswhere PS-2, PS-3, PS-4 and PS-5 were used, respectively, and one eachclone having one molecule of the double-stranded DNA fragment encoding(Aβ4-10)×3 in a state where the fragment was correctly inserted in theforward direction was obtained. The probability with which a modifiedA1aB1b gene wherein the fragment was correctly inserted can be obtainedvaried among the insertion sites, and insertion of the fragment wasespecially difficult in the case where PS-3 was used.

All of the thus prepared genes encoding modified A1aB1b were subjectedto confirmation of their nucleotide sequences. Unless otherwisespecified, the hereinafter-mentioned determination of nucleotidesequences was carried out using the DNA sequencing service by FASMACCo., Ltd.

Subsequently, in order to prepare the modified A1aB1b genes wherein DNAsencoding (Aβ4-10)×3 are inserted in plural variable regions, PCR wascarried out using the above-prepared genes encoding modified A1aB1b astemplates and the primer sets that were the same as described above.Ligation reaction of the obtained DNA fragment and the double-strandedDNA fragment encoding the above-described (Aβ4-10)×3 peptide wasrepeated, to prepare the genes encoding modified A1aB1b. By thisprocess, plasmids containing the genes encoding modified A1aB1b, inwhich DNAs encoding (Aβ4-10)×3 are inserted in plural variable regionsof the A1aB1b gene, were prepared. The particular insertion regions, andthe names of the genes encoding modified A1aB1b corresponding theretoare shown in Table 1.

TABLE 1 Region of insertion of DNA encoding Name (Aβ4-10) × 3, andnumber of inserted DNA A1aB1bM1 one each in PS-1 region, PS-2 region andPS-4 region A1aB1bM2 one in PS-1 region A1aB1bM3 one in PS-2 regionA1aB1bM4-1 one in PS-3 region A1aB1bM4-2 one in PS-4 region A1aB1bM5 oneeach in PS-1 region, PS-2 region, PS-3 region, PS-4 region and PS-5region

To allow soybean seed-specific expression of the genes encoding modifiedA1aB1b prepared as described above, the promoter region and theterminator region of the wild-type A1aB1b gene were isolated.

Using, as a probe, the promoter region of the known partial genomesequence of the A1aB1b gene (GenBank accession No. X15121) containing apartial promoter sequence of the A1aB1b gene of 639 bp, a MisuzudaizuTAC library (www.kazusa.or.jp/ja/plant/PG2HP Transformation competentbacterial artificial chromosome) kept in National Agricultural ResearchCenter for Hokkaido Region, National Agriculture and Food ResearchOrganization was screened. As a result of analysis of the nucleotidesequence of the obtained clone, it was revealed that the promoter regionlocated 2202 bp upstream of the translation initiation site of theA1aB1b gene was contained in the clone. Based on the obtained nucleotidesequence, a primer set composed of the oligonucleotide pair of SEQ IDNOs:16 and 17 was prepared, which primer set was then used for carryingout PCR in order to isolate the promoter region.

The above PCR was performed using the genomic DNA of Misuzudaizu as atemplate and 50 μL/reaction of a reaction solution, by carrying out 1cycle of 2 minutes of denaturation at 94° C. and then 25 cycles of 30seconds of denaturation at 94° C., 30 seconds of annealing at 57° C. and2 minutes 30 seconds of extension at 68° C. By this process, a promoterfragment of the A1aB1b gene having a length of 2202 bp (Gy1P) (SEQ IDNO:18) was obtained.

Subsequently, in order to isolate the terminator region of the A1aB1bgene, a primer set composed of SEQ ID NOs:19 and 20 was prepared basedon the known partial genomic sequence of the wild-type A1aB1b gene(GenBank accession No. X53404), which primer set was then used forcarrying out PCR.

The above PCR was performed using the genomic DNA of Misuzudaizu as atemplate and 50 μL/reaction of a reaction solution, by carrying out 1cycle of 2 minutes of denaturation at 94° C. and then 25 cycles of 30seconds of denaturation at 94° C., 30 seconds of annealing at 57° C. and1 minute of extension at 68° C. By this process, a terminator fragmentof the wild-type A1aB1b gene having a length of 1052 bp (Gy1T) (SEQ IDNO:21) was obtained.

To allow seed-specific expression of the genes encoding modified A1aB1b,each of the genes encoding modified A1aB1b, and Gy1P and Gy1T, whichwere obtained as described above, were ligated with the known pUHGvector (Y. Kita, K. Nishizawa, M Takahashi, M. Kitayama, M. Ishimoto.(2007) Genetic improvement of somatic embryogenesis and regeneration insoybean and transformation of the improved breeding lines. Plant CellReports 26:439-447) (FIG. 3) to construct an expression plasmid.

In order to obtain DNA fragments encoding 5 types of modified A1aB1bamong those described above, PCR was carried out using theabove-described A1aB1bM2 (SEQ ID NO:22), A1aB1bM3 (SEQ ID NO:24),A1aB1bM4-1 (SEQ ID NO:26), A1aB1bM1 (SEQ ID NO:28) and A1aB1bM5 (SEQ IDNO:30) as templates, and the primer set composed of the oligonucleotidepair of SEQ ID NOs:32 and 33.

The above PCR was performed using 50 μL/reaction of a reaction solution,by carrying out 1 cycle of 2 minutes of denaturation at 94° C. and then25 cycles of 30 seconds of denaturation at 94° C., 30 seconds ofannealing at 57° C. and 2 minutes of extension at 68° C. By thisprocess, a DNA fragment of each modified A1aB1b gene was obtained.

The DNA fragment of the modified A1aB1b gene, the promoter DNA fragmentand the terminator DNA fragment were subjected to phosphorylationreaction, and then ligated into pUHG vector which had been preliminarilydigested with SmaI and dephosphorylated with CIAP (manufactured byTAKARA BIO INC.). By analyzing the nucleotide sequences of the obtainedclones, clones in which the Gy1P promoter, the gene encoding modifiedA1aB1b and the Gy1T terminator are correctly linked in this order wereselected.

By this process, the following five types of plant transformationvectors (pUHG A1aB1bM1, pUHGA1aB1bM2, pUHGA1aB1bM3, pUHGA1aB1bM4-1 andpUHGA1aB1bM5) that express the genes encoding modified A1aB1b in aseed-specific manner were constructed.

Example 2 Construction of Expression Plasmid for Modified Common BeanArcelin

Expression plasmids for expression, in soybean seeds, of genes encodingmodified arcelin in which two copies of Aβ4-10 tandemly linked to eachother (hereinafter abbreviated as (Aβ4-10)×2) are inserted wereconstructed.

Since the variable region(s) of the gene encoding the known common beanarcelin 5-1 (GenBank accession No. Z50202) (SEQ ID NO:36) has/have notbeen revealed, assumption of the variable region(s) was carried out.Based on assumption of the variable region(s) by comparison of the DNAsequence with that of A1aB1b, it was revealed that the disorder regionis restricted to the C terminus. Therefore, it was thought that thepeptide sequence may be inserted into the C terminus. In order tofurther specify the variable region, the DNA sequence of the gene wascompared with that of phytohemagglutinin, which belongs to 2S albumin asarcelin does, and it was revealed that the loop structure of 8 to 10residues found in phytohemagglutinin is absent in the downstream of thelysine (amino acid position 149 in SEQ ID NO:37), which is thecorresponding portion in Arc5-1. Therefore, the nucleotide sequenceregion in SEQ ID NO:36 that encodes this portion (amino acid positions149-150) was assumed to be the variable region A. Subsequently, arcelin1 was compared with one of the storage proteins of common bean,phaseolin. As a result, a gap of 7 residues was found in the downstreamof asparagine corresponding to amino acid position 250 of SEQ ID NO:37.Therefore, the nucleotide sequence region encoding this portion (aminoacid positions 250-251) was assumed to be the variable region B.

In order to incorporate the DNA encoding (Aβ4-10)×2 into the assumedvariable regions of the gene encoding Arc5-1, the oligonucleotideencoding (Aβ4-10)×2 (420F, SEQ ID NO:34) and the oligonucleotidecomplementary thereto (420R, SEQ ID NO:35) were synthesized.

In the presence of ATP at a final concentration of 1 mM, 100 pmol eachof 420F and 420R was subjected to phosphorylation reaction with T4Polynucleotide Kinase (manufactured by TAKARA BIO INC.), and thereaction solutions after the reaction were mixed together, followed byheating the resulting mixture at 94° C. for 10 minutes and then allowingthe mixture to cool gradually to 37° C. for 1 hour, thereby carrying outannealing. By this process, a double-stranded DNA fragment encoding(Aβ4-10)×2 was obtained.

Using, as a template, the plasmid pBSK-Arc5-1 (kept in NationalAgricultural Research Center for Hokkaido Region, National Agricultureand Food Research Organization) obtained by inserting cDNA encodingArc5-1 into the SmaI site of pBluescript II SK(−) (manufactured byStratagene), PCR was carried out to obtain a DNA fragment containing thevector portion, such that the amino acid portion of a particularvariable region of the gene encoding Arc5-1 is positioned at the ends.This DNA fragment was ligated with the double-stranded DNA fragmentencoding (Aβ4-10)×2 synthesized as described above, to prepare a plasmidcontaining the gene encoding modified Arc5-1.

The method is more concretely described below.

A total of two primer sets, that is, the primer set (PS-A) composed ofthe primer pair of SEQ ID NOs:38 and 39 for insertion into the variableregion A, and the primer set (PS-B) composed of the primer pair of SEQID NOs:40 and 41 for insertion into the variable region B, in the geneencoding Arc5-1 were prepared. The primers were synthesized such thatnucleotide substitutions for introducing amino acid substitutions in theimmediate upstream and downstream of the insertion regions wereintroduced in order to allow cleaving out of Aβ4-10 from the modifiedArc5-1 proteins using a protease thermolysin.

The regions in the nucleotide sequence of SEQ ID NO:36, into which theDNA encoding (Aβ4-10)×2 was inserted using the respective primer setsare hereinafter referred to as the PS-A region and PS-B region,respectively.

PCR was carried out using 10 ng of pBSK-Arc5-1 as a template. This PCRwas performed using 50 μL/reaction of a reaction solution, by carryingout 1 cycle of 2 minutes of denaturation at 94° C. and then 25 cycles of30 seconds of denaturation at 94° C., 30 seconds of annealing at 57° C.and 4 minutes of extension at 68° C. The thus obtained DNA fragment andthe double-stranded DNA fragment encoding the above-described (Aβ4-10)×2were subjected to ligation reaction.

By this process, 2 types of plasmids containing a gene encoding modifiedArc5-1, wherein the DNA encoding (Aβ4-10)×2 is inserted in a variableregion of the gene encoding Arc5-1, were prepared. The particularinsertion regions of the DNAs encoding (Aβ4-10)×2, and the names of thegenes encoding modified Arc5-1 corresponding thereto are shown in Table2.

TABLE 2 Region of insertion of DNA encoding Name (Aβ4-10) × 2, andnumber of inserted DNA Arc5M1 one in PS-A region Arc5M2 one in PS-Bregion

In order to allow soybean seed-specific expression of modified Arc5-1,each of the above obtained Arc5M1 and Arc5M2, and Gy1P and Gy1T derivedfrom the A1aB1b gene in the above Example 1, were ligated with the pUHGvector to construct an expression plasmid. In order to obtain DNAfragments encoding modified Arc5-1, PCR was carried out using theabove-described Arc5M1 and Arc5M2 as templates, and the primer setcomposed of the oligonucleotide pair of SEQ ID NOs:42 and 43.

The above PCR was performed using 50 μL/reaction of a reaction solution,by carrying out 1 cycle of 2 minutes of denaturation at 94° C. and then25 cycles of 30 seconds of denaturation at 94° C., 30 seconds ofannealing at 57° C. and 1 minute of extension at 68° C. By this process,a DNA fragment of each modified Arc5-1 gene was obtained.

The DNA fragment of the modified Arc5-1 gene, Gy1P and Gy1T weresubjected to phosphorylation reaction, and then ligated into the pUHGvector which had been preliminarily digested with SmaI anddephosphorylated.

By this process, plant transformation vectors pUHG Arc5M1 and pUHGArc5M2 that express the modified Arc5-1 genes in a seed-specific mannerwere constructed.

Example 3 Construction of Expression Plasmid for Modified Rice Prolamin

An expression plasmid for expression, in soybean seeds, of a geneencoding modified prolamin in which DNA encoding (Aβ4-10)×2 is insertedwas constructed.

Since the variable region(s) of the gene encoding the known riceprolamin 10K, RP10 (GenBank accession No. E09782) (SEQ ID NO:44),has/have not been revealed, assumption of the variable region(s) wascarried out. The amino acid sequence of RP10 was compared with that ofzein delta, which is one of the major storage proteins in maize. As aresult, a gap of 11 residues was found in the downstream of lysinecorresponding to amino acid position 110 of SEQ ID NO:45. Therefore, thenucleotide sequence region encoding this portion (amino acid positions110-111) was assumed to be the variable region a.

Subsequently, using, as a template, the plasmid pBSK-RP10 (kept inNational Agricultural Research Center for Hokkaido Region, NationalAgriculture and Food Research Organization) obtained by cloning the cDNAencoding the rice prolamin 10K, RP10, into the SmaI site of pBluescriptII SK(−) (manufactured by Stratagene), PCR was carried out to obtain aDNA fragment containing the vector portion, such that the amino acidportion of the particular variable region a of the gene encoding RP10 ispositioned at the ends. This DNA fragment was ligated with thedouble-stranded DNA fragment encoding (Aβ4-10)×2 synthesized in Example2, to prepare a plasmid containing a gene encoding modified RP10.

The method is more concretely described below.

A primer set composed of the primer pair of SEQ ID NOs:46 and 47 forinsertion into the variable region a in the gene encoding RP10 wasprepared. The primers were synthesized such that nucleotidesubstitutions for introducing amino acid substitutions in the immediateupstream and downstream of the insertion region were introduced in orderto allow cleaving out of the Aβ4-10 peptide from the modified RP10protein using a protease thermolysin.

PCR was carried out using 10 ng of pBSK-RP10 as a template. This PCR wasperformed using 50 μL/reaction of a reaction solution, by carrying out 1cycle of 2 minutes of denaturation at 94° C. and then 25 cycles of 30seconds of denaturation at 94° C., 30 seconds of annealing at 57° C. and4 minutes of extension at 68° C. Each of the obtained DNA fragments andthe double-stranded DNA fragment encoding the above-described (Aβ4-10)×2were subjected to ligation reaction.

By this process, a plasmid (RP10M1) containing a gene encoding modifiedRP10, wherein the DNA encoding (Aβ4-10)×2 is inserted in the variableregion of the gene encoding RP10, was prepared.

In order to allow seed-specific expression of the gene encoding modifiedRP10, the gene encoding modified RP10, and Gy1P and Gy1T obtained in theabove Example 1 were ligated with the pUHG vector to construct anexpression plasmid. In order to obtain a DNA fragment encoding modifiedRP10, PCR was carried out using the above-described RP10M1 as atemplate, and the primer set composed of SEQ ID NOs:48 and 49.

The above PCR was performed using 50 μL /reaction of a reactionsolution, by carrying out 1 cycle of 2 minutes of denaturation at 94° C.and then 25 cycles of 30 seconds of denaturation at 94° C., 30 secondsof annealing at 57° C. and 1 minute of extension at 68° C. By thisprocess, a DNA fragment encoding modified RP10 was obtained.

The DNA fragment encoding modified RP10, and the promoter DNA fragmentand the terminator DNA fragment were subjected to phosphorylationreaction, and then ligated into the pUHG vector which had beenpreliminarily digested with SmaI and dephosphorylated.

By this process, a plant transformation vector pUHG RP10M1 thatexpresses the gene encoding modified RP10 in a seed-specific manner wasconstructed.

Example 4 Construction of Expression Plasmids for Respective ModifiedTypes Using Arcelin 2 Promoter

Expression plasmids for expressing, in soybean seeds, the genes encodingmodified A1aB1b prepared in Example 1 with a common bean-derived arcelin2 promoter were constructed.

(1) Isolation of Common Bean-Derived Arcelin 2 Promoter

From 1 g of fresh leaves of the wild species of common bean (linenumber: G12866), 50 μg of genomic DNA was extracted using DNeasy PlantMaxi kit (manufactured by QIAGEN).

After digesting 280 ng of the genomic DNA with the restriction enzymeSauIIIAI, dGTP was added to the resulting digestion product, followed bycarrying out single-nucleotide extension reaction (the first extensionreaction) using klenow enzyme (manufactured by Promega KK). Thereafter,the reaction product was ligated with the RWA-1 adapter included inRightWalk Kit™ using Ligation high (manufactured by Toyobo Co., Ltd.),and the resulting ligation product was used as a template for PCR toisolate the DNA in the upstream region of the arcelin 2 gene.

Subsequently, based on the nucleotide sequence of cDNA of the knowncommon bean arcelin 2 gene (GenBank accession No. M28470),oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 50and 51 (which were designated the primer SP1 and the primer SP2,respectively) were prepared using the custom synthesis service by FASMACCo., Ltd.

SEQ ID NO: 50 (primer SP1) TTGGTTTTGT TGAACGTCTC GAC SEQ ID NO: 51(primer SP2) GGTGAGAAGC ACAAGGAAGA GG

Thereafter, PCR was carried out using, as a template, 2.8 ng of theabove-constructed genomic DNA to which the adapter was ligated, theprimer WP-1 included in RightWalk Kit™ and the primer SP1. The above PCRwas performed using 50 μL/reaction of a reaction solution, by carryingout 1 cycle of 2 minutes of denaturation at 94° C. and then 35 cycles of30 seconds of denaturation at 94° C., 30 seconds of annealing at 65° C.and 5 minutes of extension at 68° C. The reaction solution contained 200μM dNTP mixture, 1.5 mM MgSO₄ solution, each of the above primers at aconcentration of 1 μM, and KOD-Plus-Ver.2 buffer containing 1 unit ofKOD-Plus-(manufactured by Toyobo Co. Ltd.).

Thereafter, the reaction solution was 100-fold diluted, and the secondPCR was carried out using 1 μL of the resulting dilution as a template,the primer WP-2 included in RightWalk Kit™ and the primer SP2. Thecomposition of the solution and the temperature conditions in the secondPCR were the same as those in the first PCR except for the template andthe primers.

The amplified DNA fragment was subjected to phosphorylation reactionwith T4 Polynucleotide Kinase (manufactured by TAKARA BIO INC.) in thepresence of ATP at a final concentration of 1 mM, and then ligated withpBluescriptII SK(−) (manufactured by Stratagene) that had beenpreliminarily treated with SmaI.

This reaction product was designated Arc2P(i), and its nucleotidesequence was determined using the DNA sequencing service by FASMAC Co.,Ltd. As a result, it was confirmed that the product contains a novelregion having a length of 844 bp in the upstream of the initiation codonof the arcelin 2 gene.

Thereafter, in order to isolate the region located further upstream, newprimers were prepared to carry out the second elongation reaction.

After digesting 280 ng of the genomic DNA with the restriction enzymeBglII, dGTP was added to the resulting digestion product, followed bycarrying out single-nucleotide extension reaction using klenow enzyme(manufactured by Promega KK). Thereafter, the reaction product wasligated with the RWA-1 adapter included in RightWalk Kit™, and theresulting ligation product was used as a template for PCR to isolate thepromoter.

Subsequently, based on the nucleotide sequence of cDNA of the knowncommon bean arcelin 2 gene (GenBank accession No. M28470), anoligonucleotide having the nucleotide sequence shown in SEQ ID NO:52(which was designated the primer secondSP1) was prepared, and, based onthe nucleotide sequence located in a region of 844 bp upstream of theinitiation codon, which was obtained in the first extension reaction, anoligonucleotide having the nucleotide sequence shown in SEQ ID NO:53(which was designated the primer secondSP2) was prepared.

SEQ ID NO: 52 (primer secondSP1) CAGATTTTTT GCCCTCAAAA TTGATGSEQ ID NO: 53 (primer secondSP2) CGGATGTGCG TGGACTACAA GG

Thereafter, PCR was carried out using, as a template, 2.8 ng of theabove-constructed genomic DNA to which the adapter was ligated, theprimer WP-1 included in RightWalk Kit™ and the primer secondSP1. Thecomposition of the solution and the temperature conditions in the PCRwere the same as those in the above-described first extension reactionexcept for the template and the primers.

Thereafter, the above PCR solution was 100-fold diluted, and the secondPCR was carried out using 1 μL of the resulting dilution as a template,the primer WP-2 included in RightWalk Kit™ and the primer secondSP2. Thecomposition of the solution and the temperature conditions in the secondPCR were the same as those in the first PCR except for the template andthe primers.

The amplified DNA fragment was subjected to phosphorylation reactionwith T4 Polynucleotide Kinase (manufactured by TAKARA BIO INC.) in thepresence of ATP at a final concentration of 1 mM, and then ligated withpBluescriptII SK(−) (manufactured by Stratagene) that had beenpreliminarily treated with SmaI.

This reaction product was designated Arc2P(ii), and its nucleotidesequence was determined. Thereafter, in order to isolate the regionlocated further upstream, new primers were prepared to carry out thethird elongation reaction.

After digesting 280 ng of the genomic DNA with the restriction enzymeXbaI, dCTP was added to the resulting digestion product, followed bycarrying out single-nucleotide extension reaction using klenow enzyme(manufactured by Promega KK). Thereafter, the reaction product wasligated with the RWA-2 adapter included in RightWalk Kit™, and theresulting ligation product was used as a template for PCR to isolate thepromoter.

Subsequently, based on the nucleotide sequence of 197 bp obtained in thesecond extension reaction, oligonucleotides having the nucleotidesequences shown in SEQ ID NOs: 54 and 55 (which were designated theprimer thirdSP1 and the primer thirdSP2, respectively) were prepared.

SEQ ID NO: 54 (primer thirdSP1) CGACCTGAAG AACGCAGCGG CGACCSEQ ID NO: 55 (primer thirdSP2) TACCAGCAGT TGATGGACAA GATC

Thereafter, PCR was carried out using, as a template, 2.8 ng of theabove-constructed genomic DNA to which the adapter was ligated, theprimer WP-1 included in RightWalk Kit™ and the primer thirdSP1. Thecomposition of the solution and the temperature conditions in the PCRwere the same as those in the above-described first extension reactionexcept for the template and the primers.

Thereafter, the above PCR solution was 100-fold diluted, and the secondPCR was carried out using 1 μL of the resulting dilution as a template,the primer WP-2 included in RightWalk Kit™ and the primer thirdSP2. Thecomposition of the solution and the temperature conditions in the secondPCR were the same as those in the first PCR except for the template andthe primers.

The amplified DNA fragment was subjected to phosphorylation reactionwith T4 Polynucleotide Kinase (manufactured by TAKARA BIO INC.) in thepresence of ATP at a final concentration of 1 mM, and then ligated withpBluescriptII SK(−) (manufactured by Stratagene) that had beenpreliminarily treated with SmaI.

This reaction product was designated Arc2P(iii), and its nucleotidesequence was determined. As a result it was confirmed that the productcontains a novel region having a length of 2819 bp in the upstream ofArc2P(ii) (3860 bp in total). Thus, by the three times of extensionreaction, DNA(Arc2P) having a length of 3860 bp which contains the5′-untranslated region in the upstream of the initiation codon of thearcelin 2 gene, wherein the novel promoter sequence is included, wasobtained (SEQ ID NO:56, in which the promoter region corresponds tonucleotide positions 1399-3860).

(2) Isolation of Common Bean-Derived Arcelin 2 Terminator

After digesting 280 ng of the genomic DNA extracted in the above (1)with the restriction enzyme NheI, dCTP was added to the resultingdigestion product, followed by carrying out single-nucleotide extensionreaction using klenow enzyme (manufactured by Promega KK). Thereafter,the reaction product was ligated with the RWA-2 adapter included inRightWalk Kit™ using Ligation high (manufactured by Toyobo Co., Ltd.),and the resulting ligation product was used as a template for PCR toisolate the terminator gene.

Subsequently, based on the nucleotide sequence of cDNA of the knowncommon bean arcelin 2 gene (GenBank accession No. M28470),oligonucleotides having the nucleotide sequences shown in SEQ ID NOs:57and 58 (which were designated the primer SP3 and the primer SP4,respectively) were prepared.

SEQ ID NO: 57 (primer SP3) CATCAATTTT GAGGGCAAAA AATCTG SEQ ID NO: 58(primer SP4) CGTTCCAACA TCCTCCTCAA CAAGATC

Thereafter, PCR was carried out using, as a template, 2.8 ng of theabove-constructed genomic DNA to which the adapter was ligated, theprimer WP-1 included in RightWalk Kit™ and the primer SP3. The above PCRwas performed using 50 gL/reaction of a reaction solution, by carryingout 1 cycle of 2 minutes of denaturation at 94° C. and then 35 cycles of30 seconds of denaturation at 94° C., 30 seconds of annealing at 65° C.and 5 minutes of extension at 68° C. The reaction solution contained 200μM dNTP mixture, 1.5 mM MgSO₄ solution, each of the above primers at aconcentration of 1 μM, and KOD-Plus-Ver.2 buffer containing 1 unit ofKOD-Plus-(manufactured by Toyobo Co. Ltd.).

Thereafter, the reaction solution was 100-fold diluted, and the secondPCR was carried out using 1 μL of the resulting dilution as a template,the primer WP-2 included in RightWalk Kit™ and the primer SP4. Thecomposition of the solution and the temperature conditions in the secondPCR were the same as those in the first PCR except for the template andthe primers.

The amplified DNA fragment was subjected to phosphorylation reactionwith T4 Polynucleotide Kinase (manufactured by TAKARA BIO INC.) in thepresence of ATP at a final concentration of 1 mM, and then ligated withpBluescriptII SK(−) (manufactured by Stratagene) that had beenpreliminarily treated with SmaI.

This reaction product was designated Arc2T, and its nucleotide sequencewas determined. As a result, it was confirmed that the product containsa novel region having a length of 795 bp in the downstream of the stopcodon of the arcelin 2 gene wherein the 3′-untranslated region isincluded (SEQ ID NO:59).

(3) Construction of Various Modified Expression Plasmids

In order to express the genes encoding modified A1aB1b in seeds, thegenes encoding various modified A1aB1b obtained in Example 1, and Arc2Pand Arc2T were ligated with the known pUHG vector (mentioned above, FIG.3), to construct expression plasmids.

The DNA fragment of each modified A1aB1b gene, the promoter DNA fragmentand the terminator DNA fragment were subjected to phosphorylationreaction, and then ligated with the pUHG vector that had beenpreliminarily digested with SmaI and dephosphorylated with CIAP(manufactured by TAKARA BIO INC.). By analyzing the nucleotide sequencesof the obtained clones, clones in which the Arc2P promoter, the geneencoding modified A1aB1b and the Arc2T promoter are correctly linked inthis order were selected.

By this process, the following five types of plant transformationvectors (pUHGA2PA1aB1bM1, pUHGA2PA1aB1bM3 and pUHGA2PA1aB1bM5) thatexpress the genes encoding modified A1aB1b under the control of thearcelin 2 promoter in a seed-specific manner were constructed.

In the same manner, a plant transformation vector pUHGA2PRP10M1 thatexpresses the gene encoding the above-mentioned RP10M1 in aseed-specific manner was constructed.

Example 5 Introduction of Gene Encoding Modified Seed Storage Protein toSoybean

By the known method (K. Nishizawa, Y. Kita, M. Kitayama, M. Ishimoto.(2006) A red fluorescent protein, DsRed2, as a visual reporter fortransient expression and stable transformation in soybean. Plant CellReports 25:1355-1361), 30 adventitious embryonic masses (with diametersof not more than 3 mm) were induced from immature seeds of the soybeanvariety Jack and a mutant line deficient for 11S globulin and 7Sglobulin that are major seed storage proteins (kept in NationalAgricultural Research Center for Hokkaido Region, National Agricultureand Food Research Organization) (Y. Kita, K. Nishizawa, M Takahashi, M.Kitayama, M. Ishimoto. (2007) Genetic improvement of somaticembryogenesis and regeneration in soybean and transformation of theimproved breeding lines. Plant Cell Reports 26:439-447), and theseadventitious embryonic masses were placed in 1.5 ml tubes, followed bycarrying out the gene transfer operation by the whisker ultrasonicmethod (JP 3312867 B).

In a 1.5 ml tube, 5 mg of whiskers made of potassium titanate LS20(manufactured by Titan Kogyo, Ltd.) were placed, and the tube was leftto stand for 1 hour, followed by removing and completely distillingethanol to obtain sterile whiskers. Into the tube containing thewhiskers, 1 ml of sterile water was added, and the resulting mixture wasstirred well. The mixture of whiskers and sterile water was subjected tocentrifugation, and water as the supernatant was discarded. In such amanner, the whiskers were washed. This washing operation for thewhiskers was repeated 3 times. Thereafter, 0.5 ml of the known MS liquidmedium was added to the tube to obtain a whisker suspension.

To the tube containing the whisker suspension obtained as describedabove, the above-described 30 adventitious embryonic masses (withdiameters of not more than 3 mm) were added, and the resulting mixturewas stirred, followed by centrifuging the mixture at 1000 rpm for 10seconds to precipitate the adventitious embryonic masses and thewhiskers. The supernatant was discarded to obtain a mixture of theadventitious embryonic masses and the whiskers.

Into the tube containing the above mixture, 20 μl (20 μg) each of theexpression vectors for the modified seed storage proteins prepared inExamples 1 to 4 was added, and the resulting mixture was sufficientlymixed by shaking to obtain a uniform mixture.

Subsequently, this tube containing the uniform mixture was subjected tocentrifugation at 18000×g for 5 minutes. The mixture after thecentrifugation was mixed by shaking again. This operation was repeated 3times.

The thus obtained tube containing the adventitious embryonic masses, thewhiskers and the vector was placed in the bath of an ultrasonicgenerator such that the tube was sufficiently soaked therein. Anultrasonic wave with a frequency of 40 kHz was radiated to the tube atan intensity of 0.25 W/cm² for 1 minute. Thereafter, this mixture wasleft to stand for 10 minutes at 4° C. The mixture processed withultrasonication in such a manner was washed with the above-described MSliquid medium.

The processed adventitious embryonic masses were cultured in the knownliquid medium for growing adventitious embryos for 1 week by rotaryshaking culture (100 rpm), and then cultured in a fresh liquid mediumfor growing adventitious embryos containing hygromycin B (15 mg/l)(Roche Diagnostics, Mannheim, Germany) for 1 week. Further, theadventitious embryonic masses were cultured in a liquid medium forgrowing adventitious embryos containing 30 mg/l hygromycin B for 4 weeks(while exchanging the medium every week), and then subjected toselection culture in a liquid medium for growing adventitious embryoscontaining 45 mg/l hygromycin B for 1 week. The gene transfer wascarried out for 12 microtubes per vector.

Hygromycin-resistant adventitious embryonic masses were transferred to aliquid medium for maturation of adventitious embryos, and the culturewas continued with shaking (100 rpm) for 4 weeks to allow maturation ofthe adventitious embryos. The mature adventitious embryos were dried bybeing left to stand in a sterile Petri dish for 3 to 5 days, and thentransferred to the known solid medium for germination. After carryingout germination culture for 7 to 10 days, the embryos were transferredto the known rooting medium, thereby allowing the germinated seedlingsto grow. After the growth of roots and buds, the plants were transferredto a pot containing soil, and high humidity was maintained untilacclimation.

Example 6 Preparation of Transformed Soybean Plant to Which ModifiedSeed Storage Protein Gene Was Introduced

By such a process, 6 individuals of transformed soybean plants producedby introducing A1aB1BM1, 6 individuals of transformants produced byintroducing A1aB1bM2, 5 individuals of transformants produced byintroducing A1aB1bM3, 5 individuals of transformants produced byintroducing A1aB1bM4-1, and 9 individuals of transformants produced byintroducing A1aB1bM5, to the Jack variety were prepared. Further, 3individuals of transformed soybean plants to which Arc5M1 wasintroduced, 3 individuals of transformants to which Arc5M2 wasintroduced, and 2 individuals of transformed soybean plants to whichRP10M1 was introduced were prepared.

Further, 12 individuals of transformed soybean plants produced byintroducing A1aB1bM1, and 6 individuals of transformed soybean plantsproduced by introducing RP10M1, to the above-described mutant linedeficient for 11S globulin and 7S globulin (hereinafter referred to asthe seed storage protein-deficient variety) were prepared.

Further, 5 individuals of transformed soybean plants produced byintroducing A1aB1bM3 to the seed storage protein-deficient variety wereprepared.

Further, 8 individuals of transformants produced by introducingA2PA1aB1bM1, 33 individuals of transformants produced by introducingA2PA1aB1bM3, 32 individuals of transformants produced by introducingA2PA1aB1bM5, and 9 individuals of transformants produced by introducingA2PRP10M1, to the seed storage protein-deficient variety were prepared.

These plant bodies of transformed soybean were acclimatized to ambienthumidity, and the cultivation was continued under the conditions of10000 1× and illumination for 16 hours per day, after which seeds wereharvested from all the individuals. By such a process, seeds of thetransformed soybean plants of the T₁ generation were obtained.

Example 7 Evaluation of Amount of Aβ4-10 Accumulated in Seeds ofTransformed Soybean

Total protein was extracted from the seeds of the transformed soybeansobtained in the above Example 6, and the accumulated amount of Aβ4-10was evaluated by Western blotting using an antibody specific to Aβ4-10.For lines having large accumulated amounts, quantitative analysis wascarried out.

1) Amount of Accumulation of Aβ4-10 Expressed as Modified A1aB1b

20 μg of total protein extracted from seeds of each transformed soybeanwas separated by SDS-PAGE, and allowed to react with an antibodyspecific to Aβ4-10, followed by detection using ECL Advance WesternBlotting Detection Kit (manufactured by GE Healthcare Bio-Science KK). Achemiluminescence image was captured by LAS4000miniPR (manufactured byFUJIFILM Corporation), and quantitative analysis was carried out usingMultiGage, which is an analysis software included in the apparatus. As astandard sample for quantification, a His-Tag-linked recombinant proteinA1aB1bM1 prepared by the E. coli expression system was used.

As a result, the signal band corresponding to Aβ4-10 was confirmed foreach line of the transformed soybean seeds obtained in the above Example6, so that accumulation of Aβ4-10 was confirmed. Among the lines, thetransformed soybean seeds prepared by introducing A1aB1bM1, A1aB1bM3 andA1aB1bM5 to the Jack variety (lines No. 10-2, No. a-2 and No. 6-6) andthe transformed soybean seeds prepared by introducing A1aB1bM1 to theseed storage protein-deficient variety (line No. 16-2), in which Aβ4-10was highly accumulated, were subjected to measurement of the amounts ofaccumulation of Aβ4-10. The results are shown in Table 3.

TABLE 3 Amount of accumulated Aβ4-10 Modified gene Variety Line No.(μg/1 g seed weight) A1aB1bM1 Jack 10-2 35 A1aB1bM1 Deficient variety16-2 870 A1aB1bM3 Jack  a-2 42 A1aB1bM5 Jack  6-6 108

Further, comparison of the accumulated amounts of the modified seedstorage protein in the transformed soybeans prepared by introducingA1aB1BM1 (the gene in which three copies of Aβ4-10 are inserted) to theJack variety and to the seed storage protein-deficient variety wascarried out by Western blot analysis. The results are shown in FIG. 4(wherein the arrowheads indicate the bands corresponding to the modifiedseed storage proteins). As a result, it was confirmed that the amountsof accumulation of the modified seed storage protein in the transformantline 10-2 prepared by introducing A1aB1M1 to the Jack variety (seed Nos.1 to 3) were about 0.1 to 0.2% with respect to the total protein in theseeds, while the amounts of accumulation of the modified seed storageprotein in the transformant line 16-2 prepared by introducing A1aB1M1 tothe seed storage protein-deficient line (seed Nos. 1 to 3) were about 1to 2% with respect to the total protein in the seeds, which values wereabout 10 times higher than those in the above case.

Further, the accumulated amount of the modified seed storage protein inthe transformed soybeans prepared by introducing A1aB1BM3 (the gene inwhich a single copy of Aβ4-10 is inserted) to the seed storageprotein-deficient variety was measured by Western blot analysis. Theresults are shown in Table 4.

TABLE 4 Amount of accumulated Aβ4-10 Modified gene Variety Line No.(μg/1 g seed weight) A1aB1bM3 Deficient variety 5-2 1568 5-6 2504 7-32348

2) Amount of Accumulation of Aβ4-10 Expressed as Modified Arcelin

20 μg of total protein extracted from seeds of each transformed soybeanwas separated by SDS-PAGE, and allowed to react with an antibodyspecific to Aβ4-10, followed by Western blot detection of Aβ4-10 usingECL Advance Western Blotting Detection Kit (manufactured by GEHealthcare Bio-Science KK). As a result, the signal bands correspondingto the Aβ4-10 peptide was confirmed for the transformed soybean seeds towhich Arc5M1 was introduced (line 2-1) and the transformed soybean seedsto which Arc5M2 was introduced (line 2-2), so that accumulation ofAβ4-10 was confirmed (FIG. 5).

3) Amount of Accumulation of Aβ4-10 Expressed as Modified Prolamin

20 μg of total protein extracted from seeds of the RP10M1-transformedsoybean (lines 1-1 and 4-2) was separated by SDS-PAGE, and allowed toreact with an antibody against Aβ4-10, followed by Western blotdetection using ECL Advance Western Blotting Detection Kit (manufacturedby GE Healthcare Bio-Science KK). As a result, the signal bandscorresponding to the Aβ4-10 peptide was confirmed for the transformedsoybean seeds of the respective lines to which PR10M1 was introduced, sothat accumulation of the Aβ4-10 peptide was confirmed (FIG. 6).

Similarly, the signal bands corresponding to the Aβ4-10 peptide wasconfirmed for the seeds of the RP10M1-transformed soybean, so thataccumulation of Aβ4-10 was confirmed. The accumulated amount in thiscase was about 380 μg/g seed for the No. 1-1 line.

4) Amount of Accumulation of Aβ4-10 Expressed as Modified A1aB1b byArcelin 2 Promoter

20 μg of total protein extracted from seeds of each transformed soybeanwas separated by SDS-PAGE, and subjected to the detection in the samemanner as in 1) in Example 7.

As a result, the signal bands corresponding to Aβ4-10 was confirmed forthe respective lines of transformed soybean seeds obtained in the aboveExample 6, so that accumulation of Aβ4-10 was confirmed (FIG. 7).

Further, it was confirmed that the amount of accumulation of Aβ4-10 inthe transformed soybean seeds prepared by introduction of the gene intothe seed storage protein-deficient variety, which amount of accumulationwas assumed based on the intensity of the signal band, for A2PA1aB1bM1(line 4-6) was almost equivalent to that for A1aB1bM1 (line 7-1), andthat these amounts of accumulation were clearly larger than those forA1aB1bM1 (line 8-1) prepared by introduction of the gene into the Jackvariety (FIG. 7).

Example 8 Assay of Effect of Modified Seed Storage Protein

A1aB1bM1 prepared in the above Example 1 was expressed in E. coli by theknown method, to produce the protein.

In order to obtain the recombinant protein encoded by A1aB1bM1, the E.coli expression plasmid pETA1aB1bM1 was prepared by ligating A1aB1bM1with the pET21-d vector (manufactured by Novagen).

The above-described pETA1aB1bM1 was introduced to E. coli AD494(manufactured by Novagen) by a conventional method, and the recombinantE. coli was cultured in 50 ml of the known TB medium (supplemented withkanamycin at a final concentration of 15 mg/l and carbenicillin at afinal concentration of 50 mg/l) at 37° C. for 18 hours, followed byadding 10 ml of the culture to 1000 ml of the known LB medium(supplemented with kanamycin at a final concentration of 15 mg/l,carbenicillin at a final concentration of 50 mg/l and sodium chloride ata final concentration of 500 mM) as a production medium and carrying outculture at 37° C. for 2 hours. Thereafter, IPTG was added to a finalconcentration of 1 mM, and the recombinant E. coli was then cultured at20° C. for 48 hours. The cells of E. coli after the culture werecollected by centrifugation at 8000 rpm for 15 minutes. From thebacterial cells after the collection, the fraction of soluble proteinwas extracted using BugBuster Protein Extraction Reagent (manufacturedby Novagen). From the obtained fraction of soluble protein, therecombinant protein encoded by A1aB1bM1 (A1aB1bM1 protein) was purifiedusing Ni-NTA His-Bind Resins (manufactured by Novagen).

In physiological saline, 50 μg of A1aB1bM1 having the β-amyloidantigenic determinant (Aβ4-10) was dissolved, and the resulting solutionwas administered to Alzheimer's disease model mice (TgCRND8) of 4-weeksold five times at intervals of 1 week by subcutaneous injection (3individuals/group). A control group was prepared by expressing theunmodified gene encoding the wild-type A1aB1b in E. coli in the samemanner as described above, and administering the obtained wild-typeA1aB1b to the mice. Nine weeks after the administration, blood wascollected from the mice, and production of antibodies against Aβ4-10 wasconfirmed by the known sandwich method by ELISA. The antibody titer wasevidently higher in the group to which the A1aB1bM1 protein wasadministered compared to the group to which the A1aB1b protein wasadministered, so that the vaccine effect of the recombinant proteinencoded by A1aB1bM1 was confirmed.

Example 9 Thermal Stability of Modified Seed Storage Protein in SoybeanSeeds

The transformed soybean seeds obtained in the above Example 6 weresubjected to various heat treatments to test the thermal stability ofthe modified seed storage protein in the seeds.

1) Roasted Group of Transformed Soybean Seeds

The A1aB1bM3-transformed soybean seeds were pulverized, and 10 mg of thepulverized product was processed in an autoclave sterilization equipmentat 100° C. for 10 minutes, followed by extracting total protein by themethod described in the above Example 7 and evaluating the amount ofaccumulation of Aβ4-10 in the seeds by Western blotting using anantibody specific to Aβ4-10.

2) Water-Boiled Group of Transformed Soybean Seeds

The A1aB1bM3-transformed soybean seeds were pulverized, and 30 μl ofdistilled water was added to 10 mg of the pulverized product, and theresultant was processed in an autoclave sterilization equipment at 100°C. for 10 minutes, followed by extracting total protein by the methoddescribed in the above Example 7 and evaluating the amount ofaccumulation of Aβ4-10 in the seeds by Western blotting using anantibody specific to Aβ4-10.

3) Group in Which Extract from Transformed Soybean Seeds WasHeat-Treated

The A1aB1bM3-transformed soybean seeds were pulverized, and totalprotein was extracted by the method described in the above Example 7,followed by processing the total protein in an autoclave sterilizationequipment at 100° C. for 10 minutes. Thereafter, the amount ofaccumulation of Aβ4-10 in the seeds was evaluated by Western blottingusing an antibody specific to Aβ4-10.

As a result, the signal bands corresponding to Aβ4-10 was confirmed inthe roasted group and the water-boiled group. It was confirmed that theamounts were equivalent to that in the heat-untreated group, and hencethat the modified seed storage protein in the seeds is heat-stable (FIG.8).

Example 10 Form of β-Amyloid Antigenic Determinant

The peptide having the amino acid sequence encoding Aβ4-10 (P1), thepeptide having the amino acid sequence wherein two copies of P1 aretandemly linked (P2), and the peptide having the amino acid sequencewherein three copies of P1 are tandemly linked (P3) were synthesizedusing a custom peptide synthesis service.

P1: FRHDSGY (SEQ ID NO: 3) P2: FRHDSGY FRHDSGY (SEQ ID NO: 60)P3: FRHDSGY FRHDSGY FRHDSGY (SEQ ID NO: 61)

Subsequently, KLH-P1, KLH-P2 and KLH-P3, wherein a carrier proteinkey-limpet-hemocyanin (KLH, Mw. 1000000) is linked to the N-termini ofthe peptides P1, P2 and P3, respectively, through cysteine (Cys) as across-linker, were prepared.

In physiological saline, 50 μg each of these KLH-P1, KLH-P2 and KLH-P3was dissolved, and the resulting solution was administered to mice(BALBc) of 4-weeks old five times at intervals of 1 week by subcutaneousinjection (3 individuals/group). Nine weeks after the administration,blood was collected from the mice to collect antiserum. The obtainedantiserum was affinity-purified to prepare purified antibodies againstKLH-P1, KLH-P2 and KLH-P3.

Commercially available synthetic Aβ42 in amounts of 400 and 1000picomoles was subjected to electrophoresis by SDS-PAGE, and theabove-described purified antibodies against KLH-P1, KLH-P2 and KLH-P3were allowed to react with the Aβ42, followed by detection using ECLAdvance Western Blotting Detection Kit (manufactured by GE HealthcareBio-Science KK). The chemiluminescence image was captured byLAS4000miniPR (manufactured by FUJIFILM Corporation), and the signalintensities were compared to assume the binding capacities of theantibodies to Aβ42. As a result, it was shown that the signal intensityfor KLH-P2 was evidently stronger than the signal intensities for KLH-P1and KLH-P3, and hence that a specific antibody having a high antibodytiter against Aβ can be obtained by tandemly linking two copies of thepeptide having the amino acid sequence encoding Aβ4-10 (FIG. 9).

INDUSTRIAL APPLICABILITY

Since, by the present invention, it is possible to produce andaccumulate an Alzheimer's disease vaccine in soybean seeds as a fusionprotein with a seed storage protein such as soybean 11S globulin or 7Sglobulin, common bean arcelin, or rice prolamin, a large amount of theAlzheimer's disease vaccine can be produced and supplied for prophylaxisand therapy of Alzheimer's disease.

1. A transformed soybean plant comprising a gene encoding a modifiedseed storage protein introduced therein, wherein said gene encoding amodified seed storage protein has been obtained by inserting a geneencoding an Alzheimer's disease vaccine to a variable region(s) of agene encoding a wild-type seed storage protein such that frameshift doesnot occur, and wherein said modified seed storage protein is expressedand accumulates in a seed, wherein said Alzheimer's disease vaccine is aβ-amyloid antigenic determinant.
 2. (canceled)
 3. The transformedsoybean plant according to claim 1, wherein said β-amyloid antigenicdeterminant comprises a sequence having one to three copies of thepeptide comprising the sequence shown in SEQ ID NO:3 which are linked toeach other.
 4. The transformed soybean plant according to claim 1,wherein endogenous soybean 11S globulin and/or soybean 7S globulinis/are deficient.
 5. The transformed soybean plant according to claim 1,wherein said wild-type seed storage protein is the A1aB1b subunit ofsoybean 11S globulin, arcelin of common bean, or prolamin of rice. 6.The transformed soybean plant according to claim 5, wherein saidwild-type seed storage protein comprises the amino acid sequence shownin SEQ ID NO:2 or an amino acid sequence having an identity of not lessthan 90% to the amino acid sequence shown in SEQ ID NO:2, and thevariable region(s) to which said gene encoding an Alzheimer's diseasevaccine has been inserted comprises the region(s) encoding one or moreamino acid sequence(s) selected from the group consisting of the aminoacid sequences corresponding to amino acid positions 111-128, amino acidpositions 198-216, amino acid positions 268-315 and amino acid positions490-495 in SEQ ID NO:2.
 7. The transformed soybean plant according toclaim 5, wherein said wild-type seed storage protein comprises the aminoacid sequence shown in SEQ ID NO:37 or an amino acid sequence having anidentity of not less than 90% to the amino acid sequence shown in SEQ IDNO:37, and the variable region(s) to which said gene encoding anAlzheimer's disease vaccine has been inserted comprises the region(s)encoding the amino acid sequence(s) corresponding to amino acidpositions 149-150 and/or amino acid positions 250-251 in SEQ ID NO:37.8. The transformed soybean plant according to claim 5, wherein saidwild-type seed storage protein comprises the amino acid sequence shownin SEQ ID NO:45 or an amino acid sequence having an identity of not lessthan 90% to the amino acid sequence shown in SEQ ID NO:45, and thevariable region to which said gene encoding an Alzheimer's diseasevaccine has been inserted comprises the region encoding the amino acidsequence corresponding to amino acid positions 110-111 in SEQ ID NO:45.9. The transformed soybean plant according to claim 1, whereinexpression of said gene encoding a modified seed storage protein isregulated by a promoter of common bean arcelin 2 or a promoter of A1aB1bsubunit of soybean 11S globulin.
 10. (canceled)
 11. A seed of thetransformed soybean plant according to claim
 1. 12. (canceled)
 13. Amethod for producing an Alzheimer's disease vaccine using thetransformed soybean plant according to claim 1, wherein said Alzheimer'sdisease vaccine is produced in a seed of said transformed soybean plant.14. A vector comprising: a promoter which induces soybean seed-specificexpression; and a gene encoding a modified seed storage protein linkeddownstream of said promoter, wherein said gene encoding a modified seedstorage protein has been obtained by inserting a gene encoding anAlzheimer's disease vaccine to a variable region(s) of a gene encoding awild-type seed storage protein such that frameshift does not occur. 15.The vector according to claim 14, wherein said promoter is a promoter ofcommon bean arcelin 2 or a promoter of A1aB1b subunit of soybean 11Sglobulin.
 16. The transformed soybean plant according to claim 3,wherein endogenous soybean 11S globulin and/or soybean 7S globulinis/are deficient.
 17. The transformed soybean plant according to claim3, wherein said wild-type seed storage protein is the A1aB1b subunit ofsoybean 11S globulin, arcelin of common bean, or prolamin of rice. 18.The transformed soybean plant according to claim 4, wherein saidwild-type seed storage protein is the A1aB1b subunit of soybean 11Sglobulin, arcelin of common bean, or prolamin of rice.
 19. Thetransformed soybean plant according to claim 17, wherein said wild-typeseed storage protein comprises the amino acid sequence shown in SEQ IDNO:2 or an amino acid sequence having an identity of not less than 90%to the amino acid sequence shown in SEQ ID NO:2, and the variableregion(s) to which said gene encoding an Alzheimer's disease vaccine hasbeen inserted comprises the region(s) encoding one or more amino acidsequence(s) selected from the group consisting of the amino acidsequences corresponding to amino acid positions 111-128, amino acidpositions 198-216, amino acid positions 268-315 and amino acid positions490-495 in SEQ ID NO:2.
 20. The transformed soybean plant according toclaim 18, wherein said wild-type seed storage protein comprises theamino acid sequence shown in SEQ ID NO:2 or an amino acid sequencehaving an identity of not less than 90% to the amino acid sequence shownin SEQ ID NO:2, and the variable region(s) to which said gene encodingan Alzheimer's disease vaccine has been inserted comprises the region(s)encoding one or more amino acid sequence(s) selected from the groupconsisting of the amino acid sequences corresponding to amino acidpositions 111-128, amino acid positions 198-216, amino acid positions268-315 and amino acid positions 490-495 in SEQ ID NO:2.
 21. Thetransformed soybean plant according to claim 17, wherein said wild-typeseed storage protein comprises the amino acid sequence shown in SEQ IDNO:37 or an amino acid sequence having an identity of not less than 90%to the amino acid sequence shown in SEQ ID NO:37, and the variableregion(s) to which said gene encoding an Alzheimer's disease vaccine hasbeen inserted comprises the region(s) encoding the amino acidsequence(s) corresponding to amino acid positions 149-150 and/or aminoacid positions 250-251 in SEQ ID NO:37.
 22. The transformed soybeanplant according to claim 18, wherein said wild-type seed storage proteincomprises the amino acid sequence shown in SEQ ID NO:37 or an amino acidsequence having an identity of not less than 90% to the amino acidsequence shown in SEQ ID NO:37, and the variable region(s) to which saidgene encoding an Alzheimer's disease vaccine has been inserted comprisesthe region(s) encoding the amino acid sequence(s) corresponding to aminoacid positions 149-150 and/or amino acid positions 250-251 in SEQ IDNO:37.
 23. The transformed soybean plant according to claim 17, whereinsaid wild-type seed storage protein comprises the amino acid sequenceshown in SEQ ID NO:45 or an amino acid sequence having an identity ofnot less than 90% to the amino acid sequence shown in SEQ ID NO:45, andthe variable region to which said gene encoding an Alzheimer's diseasevaccine has been inserted comprises the region encoding the amino acidsequence corresponding to amino acid positions 110-111 in SEQ ID NO:45.24. The transformed soybean plant according to claim 18, wherein saidwild-type seed storage protein comprises the amino acid sequence shownin SEQ ID NO:45 or an amino acid sequence having an identity of not lessthan 90% to the amino acid sequence shown in SEQ ID NO:45, and thevariable region to which said gene encoding an Alzheimer's diseasevaccine has been inserted comprises the region encoding the amino acidsequence corresponding to amino acid positions 110-111 in SEQ ID NO:45.25. The transformed soybean plant according to claim 3, whereinexpression of said gene encoding a modified seed storage protein isregulated by a promoter of common bean arcelin 2 or a promoter of A1aB1bsubunit of soybean 11S globulin.
 26. The transformed soybean plantaccording to claim 4, wherein expression of said gene encoding amodified seed storage protein is regulated by a promoter of common beanarcelin 2 or a promoter of A1aB1b subunit of soybean 11S globulin. 27.The transformed soybean plant according to claim 5, wherein expressionof said gene encoding a modified seed storage protein is regulated by apromoter of common bean arcelin 2 or a promoter of A1aB1b subunit ofsoybean 11S globulin.
 28. The transformed soybean plant according toclaim 6, wherein expression of said gene encoding a modified seedstorage protein is regulated by a promoter of common bean arcelin 2 or apromoter of A1aB1b subunit of soybean 11S globulin.
 29. The transformedsoybean plant according to claim 7, wherein expression of said geneencoding a modified seed storage protein is regulated by a promoter ofcommon bean arcelin 2 or a promoter of A1aB1b subunit of soybean 11Sglobulin.
 30. The transformed soybean plant according to claim 8,wherein expression of said gene encoding a modified seed storage proteinis regulated by a promoter of common bean arcelin 2 or a promoter ofA1aB1b subunit of soybean 11S globulin.
 31. A seed of the transformedsoybean plant according to claim
 3. 32. A seed of the transformedsoybean plant according to claim
 4. 33. A seed of the transformedsoybean plant according to claim
 5. 34. A seed of the transformedsoybean plant according to claim
 6. 35. A seed of the transformedsoybean plant according to claim
 7. 36. A seed of the transformedsoybean plant according to claim
 8. 37. A seed of the transformedsoybean plant according to claim
 9. 38. A method for producing anAlzheimer's disease vaccine using the transformed soybean plantaccording to claim 3, wherein said Alzheimer's disease vaccine isproduced in a seed of said transformed soybean plant.
 39. A method forproducing an Alzheimer's disease vaccine using the transformed soybeanplant according to claim 4, wherein said Alzheimer's disease vaccine isproduced in a seed of said transformed soybean plant.
 40. A method forproducing an Alzheimer's disease vaccine using the transformed soybeanplant according to claim 5, wherein said Alzheimer's disease vaccine isproduced in a seed of said transformed soybean plant.
 41. A method forproducing an Alzheimer's disease vaccine using the transformed soybeanplant according to claim 6, wherein said Alzheimer's disease vaccine isproduced in a seed of said transformed soybean plant.
 42. A method forproducing an Alzheimer's disease vaccine using the transformed soybeanplant according to claim 7, wherein said Alzheimer's disease vaccine isproduced in a seed of said transformed soybean plant.
 43. A method forproducing an Alzheimer's disease vaccine using the transformed soybeanplant according to claim 8, wherein said Alzheimer's disease vaccine isproduced in a seed of said transformed soybean plant.
 44. A method forproducing an Alzheimer's disease vaccine using the transformed soybeanplant according to claim 9, wherein said Alzheimer's disease vaccine isproduced in a seed of said transformed soybean plant.